Pathogen recognition receptor (PRR) agonists are currently being developed and tested as adjuvants in various formulations to optimize the immunogenicity and efficacy of vaccines. Using an original in vitro approach to prime naive precursors from unfractionated human peripheral blood mononuclear cells, we assessed the influence of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a ligand for the stimulator of interferon genes (STING), on the induction of antigen-specific CD8 ⁺ T cells. We found that 2′3′-cGAMP and 3′3′-cGAMP were especially potent adjuvants in this system, driving the expansion and maturation of functionally replete antigen-specific CD8 ⁺ T cells via the induction of type I IFNs. The biological relevance of these findings was confirmed in vivo using two mouse models, in which 2′3′-cGAMP–adjuvanted vaccination elicited protective antitumor or antiviral CD8 ⁺ T cell responses. These results identify particular isoforms of cGAMP as effective adjuvants that may find utility in the development of novel immunotherapies and vaccines.

In recent years, there has been an increasing interest in immunomodulatory therapy as a means to treat various conditions, including infectious diseases. For instance, Toll-like receptor (TLR) agonists have been evaluated for treatment of genital herpes. However, although the TLR7 agonist imiquimod was shown to have antiviral activity in individual patients, no significant effects were observed in clinical trials, and the compound also exhibited significant side effects, including local inflammation. Cytosolic DNA is detected by the enzyme cyclic GMP-AMP (2’3’-cGAMP) synthase (cGAS) to stimulate antiviral pathways, mainly through induction of type I interferon (IFN)s. cGAS is activated upon DNA binding to produce the cyclic dinucleotide (CDN) 2’3’-cGAMP, which in turn binds and activates the adaptor protein Stimulator of interferon genes (STING), thus triggering type I IFN expression. In contrast to TLRs, STING is expressed broadly, including in epithelial cells. Here we report that natural and non-natural STING agonists strongly induce type I IFNs in human cells and in mice in vivo, without stimulating significant inflammatory gene expression. Systemic treatment with 2’3’-cGAMP reduced genital herpes simplex virus (HSV) 2 replication and improved the clinical outcome of infection. More importantly, local application of CDNs at the genital epithelial surface gave rise to local IFN activity, but only limited systemic responses, and this treatment conferred total protection against disease in both immunocompetent and immunocompromised mice. In direct comparison between CDNs and TLR agonists, only CDNs acted directly on epithelial cells, hence allowing a more rapid and IFN-focused immune response in the vaginal epithelium. Thus, specific activation of the STING pathway in the vagina evokes induction of the IFN system but limited inflammatory responses to allow control of HSV2 infections in vivo. Herpes simplex virus (HSV)-2 is the leading cause of genital ulcers, and HSV-2 infection has also been reported to amplify HIV-transmission. So far, all attempts at making an effective anti-HSV2 vaccine have failed. In recent years, there has been an increasing interest in immunomodulatory therapy as a means to treat infections. Although the TLR7 agonist imiquimod has been shown to have antiviral activity in individual patients, no significant effects were observed in clinical trials, and the compound also exhibited significant side effects including local inflammation. Type I interferon (IFN)s are key players in antiviral defense, and it is now known that the DNA sensor cyclic GMP-AMP synthase produces the cyclic di-nucleotide (CDN) 2’3’-cyclic GMP-AMP (cGAMP), which activates the adaptor protein STING to induce IFN expression. In this work we show that natural and non-natural CDNs activate strong type I IFN responses in vivo without stimulating significant expression of genes driven by the transcription factor NF-κB, which induces inflammation. Application of CDNs at epithelial surfaces gave rise to local IFN activity, but only limited systemic responses. Importantly, all tested treatment regimens, strongly reduced replication of HSV-2 in a model for genital herpes, and significantly reduced development of disease. Finally, when comparing to TLR agonists, CDNs showed the best profile with strong IFN response specifically in the epithelial cells and limited induction of inflammation.

TLR agonists are currently being developed and tested as adjuvants in various formulations to optimize the immunogenicity and efficacy of vaccines. The aim of this study was to evaluate the immunostimulatory properties of a novel compound incorporating covalently linked moieties designed to stimulate both TLR2 and TLR7. This dual TLR2/TLR7 agonist induced the maturation of dendritic cells and primed substantial populations of cytolytic and highly polyfunctional effector CD8⁺ T cells in vitro, and safely potentiated the immunogenic properties of a nanoparticulate Ag in vivo, eliciting humoral responses with a balanced T_H1/T_H2 profile in mice. Collectively, these data reveal the potential utility of chimeric adjuvants with synergistic activities mediated via TLRs.

Nexavant was reported as an alternative to the TLR3 agonist of Poly(I:C) and its derivatives. The physicochemical properties, signaling pathways, anti-cancer effects, and mechanisms of Nexavant were investigated. The distinctive characteristics of Nexavant compared to that of Poly(I:C) were demonstrated by precise quantification, enhanced thermostability, and increased resistance to RNase A. Unlike Poly(I:C), which activates TLR3, RIG-I, and MDA5, Nexavant stimulates signaling through TLR3 and RIG-I but not through MDA5. Compared to Poly(I:C), an intratumoral Nexavant treatment led to a unique immune response, immune cell infiltration, and suppression of tumor growth in various animal cancer models. Nexavant therapy outperformed anti-PD-1 antibody treatment in all the tested models and showed a synergistic effect in combinational therapy, especially in well-defined cold tumor models. The effect was similar to that of nivolumab in a humanized mouse model. Intranasal instillation of Nexavant led to the recruitment of immune cells (NK, CD4+ T, and CD8+ T) to the lungs, suppressing lung metastasis and improving animal survival. Our study highlighted Nexavant’s defined nature for clinical use and unique signaling pathways and its potential as a standalone anti-cancer agent or in combination with anti-PD-1 antibodies.

Broadly neutralizing antibodies (bNAbs) offer promising opportunities for preventing HIV-1 infection. The protection mechanisms of bNAbs involve the Fc domain, as well as their Fab counterpart. Here, different bNAb isotypes including IgG1, IgA1, IgA2, and IgA122 (IgA2 with the hinge of IgA1) were generated and then produced in CHO cells. Their ability to neutralize pseudovirus and primary HIV-1 isolates were measured, as well as their potential ADCC-like activity using a newly developed assay. In our work, gp41-specific IgA seems to be more efficient than IgG1 in inducing ADCC-like activity, but not in its virus neutralization effect. We show that either gp120-specific IgA or IgG1 isotypes are both efficient in neutralizing different viral strains. In contrast, gp120-specific IgG1 was a better ADCC-like inducer than IgA isotypes. These results provide new insights into the neutralization and ADCC-like activity of different bNAbs that might be taken into consideration when searching for new treatments or antibody-based vaccines.

Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, pro-inflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly, cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also cleave and inactivate the pyroptosis executioner Gasdermin (GSDM)-D. Subsequently, Caspase-3 and GSDM-E promote alternative cell pyroptosis, a process exacerbated in cells exhibiting impaired type I interferon production. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to Interferon alterations highlight GSDM-E/Caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia. Funding: This project was funded by grants to different co-authors as follows: the Fondation pour la Recherche Médicale (F.R.M.) to E.M. , the ERC StG (INFLAME) to E.M., the ERC StG (ANTIViR) to C.G., and by the French Ministry of Health with the participation of the Groupement Interrégional de Recherche Clinique et d’Innovation Sud-Ouest Outre- Mer (PHRCI 2020 IMMUNOMARK-COV) to G-M.B. The ASB3 structure is supported by LABEX, Investissement d’Avenir and foundation Bettencourt grants to O.N. M.P. and R.P. were respectively funded by a CIFRE PhD fellowship and a research grant from InvivoGen. S.B. is supported by a PhD fellowship from Mali Ministry of Education and from the FRM (FDT 12794). S.A.L.C. is supported by a Vaincre La Mucoviscidose (VLM) PhD fellowship. Declaration of Interests: Authors declare no conflict of interest. Ethics Approval Statement: Clinical data and blood samples for plasma isolation and cryopreservation were collected at the Toulouse University Hospital, France, in the frame of the COVID-BioToul biobank. All donors had given written informed consent and the study was approved by the ethical review board “Comité de Protection des Personnes Est-III” (ID-RCB 2020-A01292-37). Trial Registration: ClinicalTrials.gov Identifier: NCT04385108